Links

Research & Scholarship

Current Research and Scholarly Interests

The focus of the research program are clinical trials in breast cancer. Clinical trials aim to investigate treatments for women with breast cancer who are newly diagnosed or have had a recurrence of breast cancer. Trials include participation in the national breast cancer study groups, ECOG and NSABP, as well as the development of new chemotherapy programs. Of particular interest are adjuvant therapy, treatment of metastatic disease, methods for treatment of locally advanced breast cancer and inflammatory breast cancer. Laboratory work has centered on the mechanisms of how cells acquire specific fates during growth and development , providing insights into normal and abnormal mechanisms of growth control.

Clinical Trials

Comparison of Two Combination Chemotherapy Regimens in Treating Women With Breast CancerNot Recruiting

RATIONALE: Drugs used in chemotherapy, such as fluorouracil, epirubicin, cyclophosphamide,
and doxorubicin, work in different ways to stop tumor cells from dividing so they stop
growing or die. It is not yet known which combination chemotherapy regimen is more effective
in treating breast cancer.
PURPOSE: This randomized phase III trial is studying two combination chemotherapy regimens
to compare how well they work in treating women who have undergone surgery for breast cancer
that has not spread to the lymph nodes.

Stanford is currently not accepting patients for this trial.For more information, please contact Marilyn Florero, (650) 724 - 1953.

A Study of AC Followed by a Combination of Paclitaxel Plus Trastuzumab or Lapatinib or Both Given Before Surgery to Patients With Operable HER2 Positive Invasive Breast CancerNot Recruiting

The primary purpose of this study is to determine whether breast cancer tumors respond (as
measured by pathologic complete response: the absence of microscopic evidence of invasive
tumor cells in the breast) to combined chemotherapy of AC(doxorubicin and cyclophosphamide)
followed by paclitaxel plus trastuzumab or lapatinib or both given before surgery to
patients with HER2-positive breast cancer. Trastuzumab will also be given to all patients
after surgery. The study will also evaluate the toxic effects of the chemotherapy
combination, including effects on the heart, and will determine survival and
progression-free survival 5 years after treatment. Also, the study will look at whether
there are gene expression profiles in the tumor tissue that can predict pathologic complete
response.

Stanford is currently not accepting patients for this trial.For more information, please contact Marilyn Florero, (650) 724 - 1953.

RATIONALE: Zoledronate, clodronate, or ibandronate may delay or prevent bone metastases in
patients with nonmetastatic breast cancer. It is not yet known whether zoledronate is more
effective than clodronate or ibandronate in treating breast cancer.
PURPOSE: This randomized phase III trial is studying zoledronate to see how well it works
compared to clodronate or ibandronate in treating women who have undergone surgery for stage
I, stage II, or stage III breast cancer.

Stanford is currently not accepting patients for this trial.For more information, please contact Mary Chen, (650) 723 - 8686.

Primary Objective:
1. To provide preliminary data on the effects of armodafinil and Brief Behavioral Therapy
for Insomnia (BBT-I) (alone or in combination) on insomnia in breast cancer patients
receiving chemotherapy.
Secondary Objectives:
1. To provide preliminary data on the influence of armodafinil and BBT-I (alone or in
combination) on cancer-related fatigue (CRF) in breast cancer patients receiving
chemotherapy.
2. To provide preliminary data on the influence of armodafinil and BBT-I (alone or in
combination) on QOL in breast cancer patients receiving chemotherapy.
3. To provide preliminary data on influence of armodafinil and BBT-I (alone or in
combination) on endocrine and inflammatory physiological markers (measured by cortisol
and inflammatory cytokines markers)

Stanford is currently not accepting patients for this trial.For more information, please contact Oxana Palesh, PhD, MPH, 650-725-7011.

Chemotherapy With or Without Trastuzumab After Surgery in Treating Women With Invasive Breast CancerNot Recruiting

This randomized phase III clinical trial studies chemotherapy with or without trastuzumab
after surgery to see how well they work in treating women with invasive breast cancer. Drugs
used in chemotherapy work in different ways to stop the growth of tumor cells, either by
killing the cells, by stopping them from dividing, or by stopping them from spreading.
Giving more than one drug (combination chemotherapy) and giving chemotherapy after surgery
may kill more tumor cells. Monoclonal antibodies, such as trastuzumab, can block cancer
growth in different ways. Some block the ability of tumor cells to grow and spread. Others
find tumor cells and help kill them or carry tumor-killing substances to them. It is not yet
known whether combination chemotherapy is more effective with trastuzumab in treating breast
cancer.

Stanford is currently not accepting patients for this trial.For more information, please contact Amy Isaacson, 650-723-0501.

A Safety and Immunology Study of a Modified Vaccinia Vaccine for HER-2(+) Breast Cancer After Adjuvant TherapyNot Recruiting

The current trial, BNIT-BR-003, will evaluate the safety and biological activity of a fixed
dose of MVA-BN®-HER2 following adjuvant chemotherapy in patients with HER-2-positive breast
cancer.
The intent of vaccination is to induce a combined antibody and T-cell anti-HER-2 immune
response, which is intended to target HER-2-expressing tumor cells, and may induce tumor
regression or slow progression of disease.

Stanford is currently not accepting patients for this trial.For more information, please contact Mary Chen, (650) 723 - 8686.

This randomized phase II trial is studying how well giving gefitinib together with
anastrozole works compared to giving gefitinib together with fulvestrant in treating
postmenopausal women with recurrent or metastatic breast cancer. Estrogen can stimulate the
growth of breast cancer cells. Hormone therapy using anastrozole and fulvestrant may fight
breast cancer by blocking the use of estrogen. Gefitinib (ZD1839) may stop the growth of
cancer cells by blocking the enzymes necessary for their growth. It is not yet known whether
gefitinib is more effective when combined with anastrozole or fulvestrant in treating breast
cancer.

Stanford is currently not accepting patients for this trial.For more information, please contact Nancy Mori, (650) 724 - 0201.

RATIONALE: Drugs used in chemotherapy use different ways to stop tumor cells from dividing
so they stop growing or die. It is not yet known whether chemotherapy is effective in
treating women who have undergone surgery and radiation therapy for relapsed breast cancer.
PURPOSE: Randomized phase III trial to determine the effectiveness of adjuvant chemotherapy
in treating women who have undergone resection for local and/or regional relapsed breast
cancer.

Stanford is currently not accepting patients for this trial.For more information, please contact Marilyn Florero, (650) 724 - 1953.

Radiation Therapy (WBI Versus PBI) in Treating Women Who Have Undergone Surgery For Ductal Carcinoma In Situ or Stage I or Stage II Breast CancerNot Recruiting

RATIONALE: Radiation therapy uses high-energy x-rays to kill tumor cells. Giving radiation
therapy in different ways may kill any tumor cells that remain after surgery. It is not yet
known whether whole breast radiation therapy is more effective than partial breast radiation
therapy in treating breast cancer.
PURPOSE: This randomized phase III trial is studying whole breast radiation therapy to see
how well it works compared to partial breast radiation therapy in treating women who have
undergone surgery for ductal carcinoma in situ or stage I or stage II breast cancer.

Stanford is currently not accepting patients for this trial.For more information, please contact Marilyn Florero, (650) 724 - 1953.

Doxorubicin Hydrochloride, Cyclophosphamide, and Paclitaxel With or Without Bevacizumab in Treating Patients With Lymph Node-Positive or High-Risk, Lymph Node-Negative Breast CancerNot Recruiting

This randomized phase III trial studies doxorubicin hydrochloride, cyclophosphamide, and
paclitaxel to see how well they work with or without bevacizumab in treating patients with
cancer that has spread to the lymph nodes (lymph node-positive) or cancer that has not
spread to the lymph nodes but is at high risk for returning (high-risk, lymph node-negative
breast cancer). Drugs used in chemotherapy, such as doxorubicin hydrochloride,
cyclophosphamide, and paclitaxel, work in different ways to stop the growth of tumor cells,
either by killing the cells, by stopping them from dividing, or by stopping them from
spreading. Monoclonal antibodies, such as bevacizumab, may interfere with the ability of
tumor cells to grow and spread. Bevacizumab may also stop the growth of breast cancer by
blocking blood flow to the tumor. Giving chemotherapy after surgery may kill any tumor cells
that remain after surgery and help prevent the tumor from returning. It is not yet known
whether doxorubicin hydrochloride, cyclophosphamide, and paclitaxel are more effective with
or without bevacizumab.

Stanford is currently not accepting patients for this trial.For more information, please contact Pei-Jen Chang, (650) 725 - 0866.

Abstract

To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGFß1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.

Abstract

Trastuzumab, a monoclonal antibody targeting human epidermal growth factor receptor 2 (HER2; also known as HER-2/neu), is indicated for the treatment of women with either early stage or metastatic HER2(+) breast cancer. It kills tumor cells by several mechanisms, including antibody-dependent cellular cytotoxicity (ADCC). Strategies that enhance the activity of ADCC effectors, including NK cells, may improve the efficacy of trastuzumab. Here, we have shown that upon encountering trastuzumab-coated, HER2-overexpressing breast cancer cells, human NK cells become activated and express the costimulatory receptor CD137. CD137 activation, which was dependent on NK cell expression of the Fc?RIII receptor, occurred both in vitro and in the peripheral blood of women with HER2-expressing breast cancer after trastuzumab treatment. Stimulation of trastuzumab-activated human NK cells with an agonistic mAb specific for CD137 killed breast cancer cells (including an intrinsically trastuzumab-resistant cell line) more efficiently both in vitro and in vivo in xenotransplant models of human breast cancer, including one using a human primary breast tumor. The enhanced cytotoxicity was restricted to antibody-coated tumor cells. This sequential antibody strategy, combining a tumor-targeting antibody with a second antibody that activates the host innate immune system, may improve the therapeutic effects of antibodies against breast cancer and other HER2-expressing tumors.

Abstract

Cardiac inotropy progressively declines during diabetes mellitus. To date, the molecular mechanisms underlying this defect remain incompletely characterized. This study tests the hypothesis that ventricular myosin heavy chains (MHC) undergo carbonylation by reactive carbonyl species (RCS) during diabetes and these modifications contribute to the inotropic decline. Male Sprague-Dawley rats were injected with streptozotocin (STZ). Fourteen days later the animals were divided into two groups: one group was treated with the RCS blocker aminoguanidine for 6 weeks, while the other group received no treatment. After 8 weeks of diabetes, cardiac ejection fraction, fractional shortening, left ventricular pressure development (+dP/dt) and myocyte shortening were decreased by 9%, 16%, 34% and 18%, respectively. Ca(2+)- and Mg(2+)-actomyosin ATPase activities and peak actomyosin syneresis were also reduced by 35%, 28%, and 72%. MHC-alpha to MHC-beta ratio was 12:88. Mass spectrometry and Western blots revealed the presence of carbonyl adducts on MHC-alpha and MHC-beta. Aminoguanidine treatment did not alter MHC composition, but it blunted formation of carbonyl adducts and decreases in actomyosin Ca(2+)-sensitive ATPase activity, syneresis, myocyte shortening, cardiac ejection fraction, fractional shortening and +dP/dt induced by diabetes. From these new data it can be concluded that in addition to isozyme switching, modification of MHC by RCS also contributes to the inotropic decline seen during diabetes.

Abstract

Current practice is to perform a completion axillary lymph node dissection (ALND) for breast cancer patients with tumor-involved sentinel lymph nodes (SLNs), although fewer than half will have non-sentinel node (NSLN) metastasis. Our goal was to develop new models to quantify the risk of NSLN metastasis in SLN-positive patients and to compare predictive capabilities to another widely used model.We constructed three models to predict NSLN status: recursive partitioning with receiver operating characteristic curves (RP-ROC), boosted Classification and Regression Trees (CART), and multivariate logistic regression (MLR) informed by CART. Data were compiled from a multicenter Northern California and Oregon database of 784 patients who prospectively underwent SLN biopsy and completion ALND. We compared the predictive abilities of our best model and the Memorial Sloan-Kettering Breast Cancer Nomogram (Nomogram) in our dataset and an independent dataset from Northwestern University.285 patients had positive SLNs, of which 213 had known angiolymphatic invasion status and 171 had complete pathologic data including hormone receptor status. 264 (93%) patients had limited SLN disease (micrometastasis, 70%, or isolated tumor cells, 23%). 101 (35%) of all SLN-positive patients had tumor-involved NSLNs. Three variables (tumor size, angiolymphatic invasion, and SLN metastasis size) predicted risk in all our models. RP-ROC and boosted CART stratified patients into four risk levels. MLR informed by CART was most accurate. Using two composite predictors calculated from three variables, MLR informed by CART was more accurate than the Nomogram computed using eight predictors. In our dataset, area under ROC curve (AUC) was 0.83/0.85 for MLR (n = 213/n = 171) and 0.77 for Nomogram (n = 171). When applied to an independent dataset (n = 77), AUC was 0.74 for our model and 0.62 for Nomogram. The composite predictors in our model were the product of angiolymphatic invasion and size of SLN metastasis, and the product of tumor size and square of SLN metastasis size.We present a new model developed from a community-based SLN database that uses only three rather than eight variables to achieve higher accuracy than the Nomogram for predicting NSLN status in two different datasets.

Abstract

Women with inherited BRCA1/2 mutations are at high risk for breast cancer, which mammography often misses. Screening with contrast-enhanced breast magnetic resonance imaging (MRI) detects cancer earlier but increases costs and results in more false-positive scans.To evaluate the cost-effectiveness of screening BRCA1/2 mutation carriers with mammography plus breast MRI compared with mammography alone.A computer model that simulates the life histories of individual BRCA1/2 mutation carriers, incorporating the effects of mammographic and MRI screening was used. The accuracy of mammography and breast MRI was estimated from published data in high-risk women. Breast cancer survival in the absence of screening was based on the Surveillance, Epidemiology and End Results database of breast cancer patients diagnosed in the prescreening period (1975-1981), adjusted for the current use of adjuvant therapy. Utilization rates and costs of diagnostic and treatment interventions were based on a combination of published literature and Medicare payments for 2005.The survival benefit, incremental costs, and cost-effectiveness of MRI screening strategies, which varied by ages of starting and stopping MRI screening, were computed separately for BRCA1 and BRCA2 mutation carriers.Screening strategies that incorporate annual MRI as well as annual mammography have a cost per quality-adjusted life-year (QALY) gained ranging from less than 45,000 dollars to more than 700,000 dollars, depending on the ages selected for MRI screening and the specific BRCA mutation. Relative to screening with mammography alone, the cost per QALY gained by adding MRI from ages 35 to 54 years is 55,420 dollars for BRCA1 mutation carriers, 130,695 dollars for BRCA2 mutation carriers, and 98,454 dollars for BRCA2 mutation carriers who have mammographically dense breasts.Breast MRI screening is more cost-effective for BRCA1 than BRCA2 mutation carriers. The cost-effectiveness of adding MRI to mammography varies greatly by age.

Abstract

Development of somite cells is orchestrated by two regulatory processes. Differentiation of cells from the various somitic compartments into different anlagen and tissues is regulated by extrinsic signals from neighboring structures such as the notochord, neural tube, and surface ectoderm. Morphogenesis of these anlagen to form specific structures according to the segmental identity of each somite is specified by segment-specific positional information, based on the Hox-code. It has been shown that following experimental rotation of presomitic mesoderm or newly formed somites, paraxial mesodermal cells adapt to the altered signaling environment and differentiate according to their new orientation. In contrast, presomitic mesoderm or newly formed somites transplanted to different segmental levels keep their primordial segmental identity and form ectopic structures according to their original position. To determine whether all cells of a segment, including the dorsal and ventral compartment, share the same segmental identity, presomitic mesoderm or newly formed somites were rotated and transplanted from thoracic to cervical level. These experiments show that cells from all compartments of a segment are able to interpret extrinsic local signals correctly, but form structures according to their original positional information and maintain their original Hox expression in the new environment.

Abstract

To determine the maximum tolerated doses, toxicities, and therapeutic effect of an oral chemotherapy regimen consisting of uracil-ftorafur, etoposide, and leucovorin for metastatic breast cancer.The regimen consists of 28-day cycles of uracil-ftorafur, etoposide, and leucovorin administered orally on days 1-14. The dose of etoposide was fixed at 50 mg/m2/day, and uracil-ftorafur was escalated in 50 mg/m2/day increments from 200 to 350 mg/m2. Leucovorin, was used at a dose of 90 mg/day. Eligibility criteria required prior treatment with a taxane or anthracycline.A total of 23 patients were enrolled. Twenty patients are assessable for toxicity and 16 patients are assessable for response. All non-hematologic toxicities were grade 1 or 2. Three hematologic dose-limiting toxicities (DLTs) were observed. Partial responses were seen in 6 of 16 (37.5%, 95% confidence interval 15%, 85%) of assessable patients with durations ranging from 4 to 20 months. Stable disease was observed in 4 of 16 (25%) of patients with durations from 4 to 12 months. Median time to progression was 10.5 months. An intent to treat analysis revealed a response of 26%.The recommended dose and schedule of this combination is uracil-ftorafur 350 mg/m2, leucovorin 90 mg/day, and etoposide 50 mg/m2 for two consecutive weeks in a 4-week cycle. This all-oral regimen is well tolerated and demonstrates encouraging efficacy in a cohort of heavily pretreated patient with metastatic breast cancer.

Abstract

The first skeletal muscle fibers to form in vertebrate embryos appear in the somitic myotome. PCR analysis and in situ hybridization with isoform-specific probes reveal differences in the temporal appearance and spatial distribution of fast and slow myosin heavy chain mRNA transcripts within myotomal fibers. Embryonic fast myosin heavy chain was the first isoform expressed, followed rapidly by slow myosin heavy chains 1 and 3, with slow myosin heavy chain 2 appearing several hours later. Neonatal fast myosin heavy chain is not expressed in myotomal fibers. Although transcripts of embryonic fast myosin heavy chain were always distributed throughout the length of myotomal fibers, the mRNA for each slow myosin heavy chain isoform was initially restricted to the centrally located myotomal fiber nuclei. As development proceeded, slow myosin heavy chain transcripts spread throughout the length of myotomal fibers in order of their appearance. Explants of segments from embryos containing neural tube, notochord and somites 7-10, when incubated overnight, become innervated by motor neurons from the neural tube and express all four myosin heavy chain genes. Removal of the neural tube and/or notochord from explants prior to incubation or addition of d-tubocurare to intact explants prevented expression of slow myosin chain 2 but expression of genes encoding the other myosin heavy chain isoforms was unaffected. Thus, expression of slow myosin heavy chain 2 is dependent on functional innervation, whereas expression of embryonic fast and slow myosin heavy chain 1 and 3 are innervation independent. Implantation of sonic-hedgehog-soaked beads in vivo increased the accumulation of both fast and slow myosin heavy chain transcripts, as well as overall myotome size and individual fiber size, but had no effect on myotomal fiber phenotype. Transcripts encoding embryonic fast myosin heavy chain first appear ventrolaterally in the myotome, whereas slow myosin heavy chain transcripts first appear in fibers positioned midway between the ventrolateral and dorsomedial lips of the myotome. Therefore, models of epaxial myotome formation must account for the positioning of the oldest fibers in the more ventral-lateral region of the myotome and the youngest fibers in the dorsomedial region.

Abstract

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. It is also shown that Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation.

Abstract

Myogenesis has been a system central to investigations on mechanisms of diversification within groups of differentiating cells. Diversity among cell types has been well described in striated muscle tissue at the protein and enzymatic-function levels for decades, but it is only in recent years that some understanding of the molecular mechanisms responsible for this diversity has begun to emerge. Study of the expression of the slow isoforms of the myosin heavy chain has contributed to our understanding of how cell diversity arises within skeletal and cardiac muscle. Slow MyHc isoforms are developmentally responsive to a number of cues provided by the nervous systems, the endocrine system and, later in development, to functional demands on these developing tissues. Perhaps most informative have been studies on the mechanism for regulation of slow MyHc expression in mammals and birds where studies on the calcineurin-NF-AT pathways and nuclear hormone action have been shown to control MyHC gene expression in skeletal muscle and in the developing heart. The mechanisms involved in cell diversification in myogenesis are undoubtedly more varied and complex than those currently offered to explain cell diversification, but these recent studies have broadened our understanding of the interplay between the nervous system, the endocrine system and cell lineages in controlling cell diversification. Greater focus on the first fibers and cardiomyocytes to form in the embryo are likely to bring additional insights into the mechanism crucial for establishing the patterns of diversity required for successful formation of embryonic tissues.

Abstract

The slow myosin heavy chain 3 gene (slow MyHC3) is restricted in its expression to the atrial chambers of the heart. Understanding its regulation provides a basis for determination of the mechanisms controlling chamber-specific gene expression in heart development. The observed chamber distribution results from repression of slow MyHC3 gene expression in the ventricles. A binding site, the vitamin D response element (VDRE), for a heterodimer of vitamin D receptor (VDR) and retinoic X receptor alpha (RXR alpha) within the slow MyHC3 promoter mediates chamber-specific expression of the gene. Irx4, an Iroquois family homeobox gene whose expression is restricted to the ventricular chambers at all stages of development, inhibits AMHC1, the chick homolog of quail slow MyHC3, gene expression within developing ventricles. Repression of the slow MyHC3 gene in ventricular cardiomyocytes by Irx4 requires the VDRE. Unlike VDR and RXR alpha, Irx4 does not bind directly to the VDRE. Instead two-hybrid and co-immunoprecipitation assays show that Irx4 interacts with the RXR alpha component of the VDR/RXR alpha heterodimer and that the amino terminus of the Irx4 protein is required for its inhibitory action. These observations indicate that the mechanism of atrial chamber-specific expression requires the formation of an inhibitory protein complex composed of VDR, RXR alpha, and Irx4 that binds at the VDRE inhibiting slow MyHC3 expression in the ventricles.

Abstract

During embryonic development, and before functional innervation, a highly stereotypic pattern of slow- and fast-contracting primary muscle fibers is established within individual muscles of the limbs, from distinct populations of myoblasts. A difference between the fiber-type pattern found within chicken and quail pectoral muscles was exploited to investigate the contributions of somite-derived myogenic precursors and lateral plate-derived mesenchymal stroma to the establishment of muscle fiber-type patterns. Chimeric chicken/quail embryos were constructed by reciprocal transplantation of somites or lateral plate mesoderm at stages prior to muscle formation. Muscle fibers derived from quail myogenic precursors that had migrated into chicken stroma showed a quail pattern of mixed fast- and slow-contracting muscle fibers. Conversely, chicken myogenic precursors that had migrated into quail stroma showed a chicken pattern of nearly exclusive fast muscle fiber formation. These results demonstrate in vivo an intrinsic commitment to fiber-type on the part of the myoblast, independent of extrinsic signals it receives from the mesenchymal stroma in which it differentiates.

Abstract

A retrospective analysis of the treatment of locally advanced breast cancer (LABC) was undertaken at Stanford Medical Center to assess the outcome of patients who did not undergo surgical removal of their tumors. Between 1981 and 1998, 64 patients with locally advanced breast cancer were treated with induction chemotherapy, radiation with or without breast surgery, and additional chemotherapy. Sixty-two (97%) patients received cyclophosphamide, doxorubicin, and 5-fluorouracil (CAF) induction chemotherapy. Induction chemotherapy was followed by local radiotherapy in 59 (92%) patients. Based on the clinical response to chemotherapy and patient preference, 44 (69%) patients received no local breast surgery. Radiotherapy was followed by an additional, non-doxorubicin-containing chemotherapy in all patients. The mean age of patients was 49 years. Of the 65 locally advanced breast cancers in 64 patients, 26 (41%) were stage IIIA, 35 (55%) were stage IIIB, and 4 (6%) were stage IV (supraclavicular lymph nodes only). Response to induction chemotherapy was seen in 59 patients (92%), with 29 (45%) achieving a complete clinical response and 30 (47%) a partial clinical response. With a mean follow-up of 51 months (range 7-187 months), 43 patients (67.2%) have no evidence of recurrent disease. Eight (12.5%) have recurred locally, and 21 (32.8%) have recurred with distant metastasis. Actuarial 5-year survival is 75%, disease-free survival is 58%, and local control rate is 87.5%. These data indicate that the routine inclusion of breast surgery in a combined modality treatment program for LABC does not appear necessary for the majority of patients who experience a response to induction chemotherapy.

Abstract

Much of our understanding of early vertebrate embryogenesis derives from experimental work done with the chick embryo. Studies of the avian somite have played a key role in elucidating the developmental history of this important structure, the source of most muscle and bone in the organism. Here we review the development of the avian somite including morphological and molecular data on the origin of paraxial mesoderm, maturation of the segmental plate, specification and formation of somite compartments, and somite cell differentiation into cartilage and skeletal muscle.

Abstract

Primary skeletal muscle fibers first form in the segmented portions of paraxial mesoderm called somites. Although the neural tube and notochord are recognized as crucial in patterning myogenic cell lineages during avian and mammalian somitic myogenesis, the source, identities, and actions of the signals governing this process remain controversial. It has been shown that signals emanating from the ventral neural tube and/or notochord alone or Shh alone serve to activate MyoD expression in somites. However, beyond a role in initiating MyoD expression, little is known about the effects of Shh on primary muscle fiber formation in somites of higher vertebrates. The studies reported here investigate how the ventral neural tube promotes myogenesis and compare the effects of the ventral neural tube with those of purified Shh protein on fiber formation in somites. We show that purified Shh protein mimics actions of the ventral neural tube on somites including initiation of muscle fiber formation, enhancement of numbers of primary muscle fibers, and particularly, the formation of primary fibers that express slow myosin. There is a marked increase in slow myosin expression in fibers in response to Shh as somites mature. The effects of ventral neural tube on fiber formation can be blocked by disrupting the Shh signaling pathway by increasing the activity of somitic cyclic AMP-dependent protein kinase A. Furthermore, it was demonstrated that apoptosis is a dominant fate of somite cells, but not somitic muscle fibers, when cultured in the absence of the neural tube, and that application of Shh protein to somites reduced apoptosis. The block to apoptosis by Shh is a manifestation of the maturity of the somite with a progressive increase in the block as somites are displaced rostrally from somite III forward. We conclude that purified Shh protein in mimicking the effects of the ventral neural tube on segmented mesoderm can exert pleiotropic effects during primary myogenesis, including: control of the proliferative expansion of myogenic progenitor cells, antagonism of cell death pathways within the precursors to muscle fibers, and during the crucial process of primary myogenesis, can exert an effect on diversification of muscle fiber types.

Abstract

To study the specification of inflow structures in the heart we generated transgenic animals harboring the human alkaline phosphatase (HAP) gene driven by the proximal 840 bp of a quail SMyHC3 promoter. In transgenic mice, the SMyHC3-HAP reporter was expressed in posterior heart precursors at 8.25 dpc, in sinus venosa and in the atrium at 8.5 and 9.0 dpc, and in the atria from 10.5 dpc onwards. SMyHC3-HAP transgene expression overlapped synthesis and endogenous response to retinoic acid (RA) in the heart, as determined by antibodies directed against a key RA synthetic enzyme and by staining of RAREhsplacZ transgenic animals. A single pulse of all-trans RA administered to pregnant mice at 7.5, but not after 8.5, dpc induced cardiac dismorphology, ranging from complete absence of outflow tract and ventricles to hearts with reduced ventricles expressing both SMyHC3-HAP and ventricular markers. Blockade of RA synthesis with disulfiram inhibited RA-induced transcription and produced hearts lacking the atrial chamber. This study defines a novel marker for atrial-restricted transcription in the developing mouse heart. It also suggests that atrial-specific gene expression is controlled by localized synthesis of RA, and that exclusion of RA from ventricular precursors is essential for correct specification of the ventricles.

Abstract

We have used the slow myosin heavy chain (MyHC) 3 gene to study the molecular mechanisms that control atrial chamber-specific gene expression. Initially, slow MyHC 3 is uniformly expressed throughout the tubular heart of the quail embryo. As cardiac development proceeds, an anterior-posterior gradient of slow MyHC 3 expression develops, culminating in atrial chamber-restricted expression of this gene following chamberization. Two cis elements within the slow MyHC 3 gene promoter, a GATA-binding motif and a vitamin D receptor (VDR)-like binding motif, control chamber-specific expression. The GATA element of the slow MyHC 3 is sufficient for expression of a heterologous reporter gene in both atrial and ventricular cardiomyocytes, and expression of GATA-4, but not Nkx2-5 or myocyte enhancer factor 2C, activates reporter gene expression in fibroblasts. Equivalent levels of GATA-binding activity were found in extracts of atrial and ventricular cardiomyocytes from embryonic chamberized hearts. These observations suggest that GATA factors positively regulate slow MyHC 3 gene expression throughout the tubular heart and subsequently in the atria. In contrast, an inhibitory activity, operating through the VDR-like element, increased in ventricular cardiomyocytes during the transition of the heart from a tubular to a chambered structure. Overexpression of the VDR, acting via the VDR-like element, duplicates the inhibitory activity in ventricular but not in atrial cardiomyocytes. These data suggest that atrial chamber-specific expression of the slow MyHC 3 gene is achieved through the VDR-like inhibitory element in ventricular cardiomyocytes at the time distinct atrial and ventricular chambers form.

Abstract

Skeletal muscle fibers express members of the myosin heavy chain (MyHC) gene family in a fiber-type-specific manner. In avian skeletal muscle it is the expression of the slow MyHC isoforms that most clearly distinguishes slow- from fast-contracting fiber types. Two hypotheses have been proposed to explain fiber-type-specific expression of distinct MyHC genes during development-an intrinsic mechanism based on the formation of different myogenic lineage(s) and an extrinsic, innervation-dependent mechanism. We developed a cell culture model system in which both mechanisms were evaluated during fetal muscle development. Myoblasts isolated from prospective fast (pectoralis major) or slow (medial adductor) fetal chick muscles formed muscle fibers in cell culture, none of which expressed slow MyHC genes. By contrast, when muscle fibers formed from myoblasts derived from the slow muscle were cocultured with neural tube, the muscle fibers expressed a slow MyHC gene, while muscle fibers formed from myoblasts of fast muscle origin continued to express only fast MyHC. Motor endplates formed on the fibers derived from myoblasts of both fast and slow muscle origin in cocultures, and slow MyHC gene expression did not occur when neuromuscular transmission or depolarization was blocked. We have cloned the slow MyHC gene that is expressed in response to innervation and identified it as the slow MyHC 2 gene, the predominant adult slow isoform. cDNAs encoding portions of the three slow myosin heavy chain genes (MyHC1, slow MyHC 2, and slow MyHC 3) were isolated. Only slow MyHC 2 mRNA was demonstrated to be abundant in the cocultures of neural tube and muscle fibers derived from myoblasts of slow muscle origin. Thus, expression of the slow MyHC 2 gene in this in vitro system indicates that formation of slow muscle fiber types is dependent on both myoblast lineage (intrinsic mechanisms) and innervation (extrinsic mechanisms), and suggests neither mechanism alone is sufficient to explain formation of muscle fibers of different types during fetal development.

Abstract

The quail slow myosin heavy chain 3 (slow MyHC 3) gene is expressed in the developing heart and in slow muscles of the developing limb. It is first expressed in the pulsatile cardiac tube in the embryo, and as the heart chamberizes its expression becomes restricted to the atria. To identify regulatory elements responsible for atrial-specific expression, the 5' upstream region of slow MyHC 3 gene was investigated. An atrial regulatory domain (ARD1) between -840 and -680 acts as an atrial cell-specific enhancer in primary cardiocyte cultures. ARD1 also specifies atrial-specific expression in vivo when the ARD1/heterologous promoter was introduced into developing chick embryos by a replication-competent retroviral vector. ARD1 is the first atrial cell-specific enhancer to be identified. Fine deletion and mutation analysis within ARD1 defined a 40-base pair vitamin D3 receptor-like element that controls atrial cell-specific expression of the slow MyHC 3 gene by inhibiting its expression in ventricular cardiocytes.

Abstract

The impact of the surgical margin status on long-term local control rates for breast cancer in women treated with lumpectomy and radiation therapy is unclear.The records of 289 women with 303 invasive breast cancers who were treated with lumpectomy and radiation therapy from 1972 to 1992 were reviewed. The surgical margin was classified as positive (transecting the inked margin), close (less than or equal to 2 mm from the margin), negative, or indeterminate, based on the initial biopsy findings and reexcision specimens, as appropriate. Various clinical and pathologic factors were analyzed as potential prognostic factors for local recurrence in addition to the margin status, including T classification, N classification, age, histologic features, and use of adjuvant therapy. The mean follow-up was 6.25 years.The actuarial probability of freedom from local recurrence for the entire group of patients at 5 and 10 years was 94% and 87%, respectively. The actuarial probability of local control at 10 years was 98% for those patients with negative surgical margins versus 82% for all others (P = 0.007). The local control rate at 10 years was 97% for patients who underwent reexcision and 84% for those who did not. Reexcision appears to convey a local control benefit for those patients with close, indeterminate, or positive initial margins, when negative final margins are attained (P = 0.0001). Final margin status was the most significant determinant of local recurrence rates in univariate analysis. By multivariate analysis, the final margin status and use of adjuvant chemotherapy were significant prognostic factors.The attainment of negative surgical margins, initially or at the time of reexcision, is the most significant predictor of local control after breast-conserving treatment with lumpectomy and radiation therapy.

Abstract

Specification of myogenesis in early chicken somites and segmental plate was studied using transfilter explant cultures to determine if myogenic specification by axial structures is mediated by cell-cell contact. Formation of muscle fibers that express myosin heavy chain was assessed in somites transfilter from neural tube and notochord. Either the neural tube or the notochord from early chick embryos (ED 2) induces myogenesis in unspecified somites (Hamburger and Hamilton (HH) stages 11-14) when these tissues are separated by a 0.2- or a 0.05-micron pore filter. The ventral neural tube is found to be a strong inducer of myogenesis, while the dorsal neural tube is found to have low inducing activity. The reduced myogenic inducing activity of the dorsal neural tube is associated with an activity that inhibits myogenic differentiation in specified somites, somites containing cells already committed to myogenesis. Only 6-8 hr of transfilter exposure to neural tube is required to initiate somite myogenesis that is sustained in the absence of neural tube. Other tissues such as specified somites, heart, or ectoderm from the same aged embryo do not induce myogenesis in unspecified somites. Removal of the prospective floor plate of the caudal neural plate at HH stage 8 or 9 does not eliminate the inducing activity of the neurectoderm on unspecified somites (HH stage 11-14). Recombination of somites with neural tube or spinal cord from progressively older embryos (ED 4-20) showed myogenesis inducing activity at all ages, though the activity waned as development proceeded. Paraxial mesoderm need not segment into somites to respond to the inducing activity of the neural tube. We conclude that induction of myogenesis in somites does not require cell-cell contact between either the neural tube or the notochord and therefore induction is mediated by diffusible factor(s); that the inducing activity is localized principally to the notochord and ventral half of the neural tube, but not necessarily to the floor plate; that the myogenic response is specific to neural tube and notochord; and that the dorsal neural tube weakly induces myogenesis and contains an activity that inhibits myogenic cells from differentiating.

Abstract

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.

Abstract

Specification of the myogenic phenotype in somites was examined in the early chick embryo using organotypic explant cultures stained with monoclonal antibodies to myosin heavy chain. It was found that myogenic specification (formation of muscle fibers in explants of somites or segmental plates cultured alone) does not occur until Hamburger and Hamilton stage 11 (12-14 somites). At this stage, only the somites in the rostral half of the embryo are myogenically specified. By Hamburger and Hamilton stage 12 (15-17 somites), the three most caudal somites were not specified to be myogenic while most or all of the more rostral somites are specified to myogenesis. Somites from older embryos (stage 13-15, 18-26 somites) showed the same pattern of myogenic specification--all but the three most caudal somites were specified. We investigated the effects of the axial structures, the notochord and neural tube, on myogenic specification. Both the notochord and neural tube were able to induce myogenesis in unspecified somites. In contrast, the neural tube, but not the notochord, was able to induce myogenesis in explants of segmental plate, a structure which is not myogenic when cultured alone. When explants of specified somites were stained with antibodies to slow or fast MyHC, it was found that myofiber diversity (fast and fast slow fibers) was established very early in development (as early as Hamburger and Hamilton stage 11). We also found fiber diversity in explants of unspecified somites (the three most caudal somites from stage 11 to 15) when they were recombined with notochord or neural tube. We conclude that myogenic specification in the embryo results in diverse fiber types and is an inductive process which is mediated by factors produced by the neural tube and notochord.

Abstract

In the limb bud of the 5-day-old avian embryo, when primary muscle fibre formation is beginning and before specific muscles appear, differences in the expression of fast and slow myosin heavy chain genes can be detected among primary fibres of the premuscle masses. Myoblasts that form colonies of fibres of specific types can be isolated from these limb buds. To assess the role of myoblast commitment in specifying fibre types during embryonic development, we cloned myoblasts of specific types from embryonic and adult muscles, transfected them with a reporter gene, and transferred them into developing limb buds. After transfer, cloned myoblasts formed fibres in the limb with the same patterns of myosin heavy chain gene expression as the fibres they formed in cell culture. These results demonstrate that initial skeletal muscle fibre type diversity during avian limb development can originate, in part, from the commitment of distinct myoblast types to the formation of specific fibre types.

Abstract

For many years the mechanisms by which skeletal muscles in higher vertebrates come to be composed of diverse fiber types distributed in distinctive patterns has interested cell and developmental biologists. The fiber composition of skeletal muscles varies from class to class and from muscle to muscle within the vertebrates. The developmental basis for these events is the subject of this review. Because an individual multinucleate vertebrate skeletal muscle fiber is formed by the fusion of many individual myoblasts, more attention, in recent times, has been directed toward the origins and differences among myoblasts, and more emphasis has been placed on the lineal relationship of myoblasts to fibers. This is a review of studies related to the concepts of myogenic cell lineage in higher vertebrate development with emphases on some of the most challenging problems of myogenesis including the embryonic origins of myogenic precursor cells, the mechanisms of fiber type diversity and patterning, the distinctions among myoblasts during myogenesis, and the current hypotheses of how a variety of factors, intrinsic and extrinsic to the myoblast, determine the definitive phenotype of a muscle fiber.

Abstract

At least three slow myosin heavy chain (MHC) isoforms were expressed in skeletal muscles of the developing chicken hindlimb, and differential expression of these slow MHC isoforms produced distinct fiber types from the outset of skeletal muscle myogenesis. Immunohistochemistry with isoform-specific monoclonal antibodies demonstrated differences in MHC content among the fibers of the dorsal and ventral premuscle masses and distinctions among fibers before splitting of the premuscle masses into individual muscles (Hamburger and Hamilton Stage 25). Immunoblot analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of myosin extracted from the hindlimb demonstrated the presence throughout development of different mobility classes of MHCs with epitopes associated with slow MHC isoforms. Immunopeptide mapping showed that one of the MHCs expressed in the embryonic limb was the same slow MHC isoform, slow MHC1 (SMHC1), that is expressed in adult slow muscles. SMHC1 was expressed in the dorsal and ventral premuscle masses, embryonic, fetal, and some neonatal and adult hindlimb muscles. In the embryo and fetus SMHC1 was expressed in future fast, as well as future slow muscles, whereas in the adult only the slow muscles retained expression of SMHC1. Those embryonic muscles destined in the adult to contain slow fibers or mixed fast/slow fibers not only expressed SMHC1, but also an additional slow MHC not previously described, designated as slow MHC3 (SMHC3). Slow MHC3 was shown by immunopeptide mapping to contain a slow MHC epitope (reactive with mAb S58) and to be structurally similar to a MHC expressed in the atria of the adult chicken heart. SMHC3 was designated as a slow MHC isoform because (i) it was expressed only in those muscles destined to be of the slow type in the adult, (ii) it was expressed only in primary fibers of muscles that subsequently are of the slow type, and (iii) it had an epitope demonstrated to be present on other slow, but not fast, isoforms of avian MHC. This study demonstrates that a difference in phenotype between fibers is established very early in the chicken embryo and is based on the fiber type-specific expression of three slow MHC isoforms.

Abstract

In this study, differences between fetal and adult myoblasts in clonal and high density culture have been used to determine when adult myoblasts can first be detected during avian development. The results indicate that avian adult myoblasts are apparent as a distinct population of myoblasts during the midfetal stage of development. Three different criteria were used to differentiate fetal and adult myoblasts and demonstrate when adult myoblasts become a major proportion of the myoblast population: (1) differences in slow myosin heavy chain 1 (MHC1) isoform expression, (2) initiation of DNA synthetic activity, and (3) average myoblast length. Fetal chicken (ED10-12) pectoralis muscle (PM) myoblasts form myotubes that express slow MHC1 after prolonged culture, while adult chicken PM myoblasts do not. Fetal avian myoblasts were active in DNA synthesis and large when first isolated, reaching peak rates of synthesis by 24 hr in culture, while adult myoblasts were inactive in DNA synthesis and small when first isolated, only reaching peak rates of DNA synthesis and size at 3 days of incubation. A dramatic decrease in the percentage of muscle colonies with fibers that expressed slow MHC1 was observed between the midfetal stage and hatching in the chicken, along with a corresponding decrease in myoblast DNA synthetic activity and average length during this same period in both the chicken and the quail. Myoblast activity and average length increased again 3-4 days posthatch and a small transient increase in the number of slow MHC1-expressing clones was also associated with the massive growth of muscle that occurs in the neonatal bird. We conclude that adult myoblasts are present as a distinct population of myoblasts at least as early as the midfetal stages of avian development.

Abstract

Following skeletal muscle injury, new fibers form from resident satellite cells which reestablish the fiber composition of the original muscle. We have used a cell culture system to analyze satellite cells isolated from adult chicken and quail pectoralis major (PM; a fast muscle) and anterior latissimus dorsi (ALD; a slow muscle) to determine if satellite cells isolated from fast or slow muscles produce one or several types of fibers when they form new fibers in vitro in the absence of innervation or a specific extracellular milieu. The types of fibers formed in satellite cell cultures were determined using immunoblotting and immunocytochemistry with monoclonal antibodies specific for avian fast and slow myosin heavy chain (MHC) isoforms. We found that satellite cells were of different types and that fast and slow muscles differed in the percentage of each type they contained. Primary satellite cells isolated from the PM formed only fast fibers, while up to 25% of those isolated from ALD formed fibers that were both fast and slow (fast/slow fibers), the remainder being fast only. Fast/slow fibers formed from chicken satellite cells expressed slow MHC1, while slow MHC2 predominated in fast/slow fibers formed from quail satellite cells. Prolonged primary culture did not alter the relative proportions of fast to fast/slow fibers in high density cultures of either chicken or quail satellite cells. No change in commitment was observed in fibers formed from chicken satellite cell progeny repeatedly subcultured at high density, while fibers formed from subcultured quail satellite cell progeny demonstrated increasing commitment to fast/slow fiber type formation. Quail satellite cells cloned from high density cultures formed colonies that demonstrated a similar change in commitment from fast to fast/slow, as did serially subcloned individual satellite cell progeny, indicating that the observed change from fast to fast/slow differentiation resulted from intrinsic changes within a satellite cell. Thus satellite cells freshly isolated from adult chicken and quail are committed to form fibers of at least two types, satellite cells of these two types are found in different proportions in fast and slow muscles, and repeated cell proliferation of quail satellite cell progeny may alter satellite cell progeny to increasingly form fibers of a single type.

Abstract

We analyzed the expression of two slow myosin heavy chain isoforms (slow MHC1 and slow MHC2) in myotubes formed from embryonic and fetal chicken myoblasts in vitro and found that the myotubes formed from myoblasts of these two developmental periods had different patterns of expression of slow MHCs. In cultures of myogenic cells from the Embryonic Day 5 (E5) hindlimb, immunoblot analysis showed that two slow MHCs with the immunological and electrophoretic properties like those of slow MHC1 and slow MHC2 were expressed on Day 3 of culture and that both slow MHCs continued to be expressed through 10 days of culture. In cultures of myogenic cells from the fetal (E12) thigh, in contrast, slow MHC1 was not expressed on Day 3 of culture, but was expressed after Day 7; slow MHC2 was never expressed by myotubes formed from fetal myoblasts. Immunocytochemistry was used to further analyze slow MHC1 and slow MHC2 expression in individual myotubes formed from embryonic and fetal myoblasts in both clonal cultures and high density, cytosine arabinoside-treated cultures. These analyses showed that (i) E5 embryonic myoblasts were of two principal types, those that formed myotubes that expressed isoforms like slow MHC1 and MHC2 throughout the life of the myotube, and those that formed myotubes that did not express slow MHCs at any time; and (ii) E12 fetal myoblasts formed myotubes that at first expressed only fast MHC but expressed both fast MHC and slow MHC1--but not slow MHC2--as culture duration was lengthened. Thus, the expression patterns of slow MHC1 and slow MHC2-like isoforms appeared to be regulated by different cellular processes in myotubes formed from embryonic and fetal myoblasts.

Abstract

A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.

Abstract

The fast alkali myosin light chains LC1f and LC3f are two contractile protein isoforms encoded for by a single gene complex. Expression of these two isoforms is dependent upon differential initiation of transcription at either of two promoters encoding unique 5' exons for isoform-specific amino termini of these light chains. Studies of protein expression have shown that the two isoforms are first expressed at different stages of development and in the case of the LC3f isoform only in fast twitch muscle fiber types. The molecular mechanisms that regulate the differential transcription of the gene complex are unknown. Experiments reported here demonstrated the direct correlation of isoform protein and mRNA expression with the undermethylation of the DNA in the promoter regions of the gene for each of the expressed myosin light chain isoforms. We find that fast and slow muscles have different patterns of undermethylation of the two promoter regions of the gene. Moreover, changes in methylation of the promoter regions were shown to occur specifically in skeletal muscle tissue, to be developmentally regulated, and to only occur in the LC3f promoter of those muscle groups that express LC3f protein.

Abstract

Monoclonal antibodies (mAbs), electrophoresis, immunoblotting, and immunohistochemistry were used to determine the molecular properties of cardiac myosin heavy chain (MHC) isoforms and the regions of the developing chicken heart in which they were expressed. Adult atria expressed three electrophoretically distinct MHCs that reacted specifically with mAbs F18, F59, or S58. During embryonic Days 2-4, when the atrial and ventricular chambers are forming, MHCs that reacted with mAbs F18, F59, or S58 were expressed in both the atria and ventricles. The atria continued to express MHCs that reacted with mAbs F18, F59, or S58 at all stages of development and in the adult. In the ventricles, expression of the MHCs reacting with these mAbs was found to be developmentally regulated. By embryonic Day 16, MHC(s) reacting with mAb F18 had disappeared from the developing ventricles, whereas MHCs reacting with S58 and F59 continued to be expressed throughout the ventricles. As development continued, MHC(s) reacting with S58 in the ventricle became restricted to expression in only the ventricular conducting system. MHC(s) reacting with F59 were expressed in both the ventricular myocytes and the ventricular conducting system throughout development and in the adult. Thus, in contrast to the embryonic chicken heart where at least three MHC isoforms were expressed in both the atria and ventricles, we found in the adult chicken heart that-at a minimum-three MHC isoforms were expressed in the atria, two MHC isoforms were expressed in the ventricular conducting system, and one MHC isoform in the ventricular myocardium. MHC isoform expression in the developing avian heart appears to be more complex than previously recognized.

Abstract

Breast cancers present as a spectrum of neoplasms. The natural histories of the neoplasms are tied to the biological properties of the neoplastic cells that compose them. We review the histological, cytological, kinetic, and other parameters that best characterize the clinical biology of breast cancer.

Abstract

Fourteen patients with locally advanced breast cancer were treated at Stanford University Medical Center with a combined modality approach. Treatment consisted of an initial 5 day course of cyclophosphamide followed by three cycles of combination chemotherapy (CAF or CMF). Patients subsequently received radiation therapy to the involved breast and regional nodal areas, followed by mastectomy if resistant disease was present following irradiation. Additional chemotherapy (CMF) was administered for 6 cycles. With a median follow up of 42 months, all fourteen patients are free of local disease. Five out of the fourteen patients have experienced distant relapses and two patients died. We conclude that an aggressive combined modality approach to treatment of locally advanced breast cancer can result in excellent local control and survival even in poor prognosis patients. A review of pertinent studies on multimodality treatment for locally advanced breast cancer confirms our findings.

Abstract

We have identified three sarcolemma-associated antigens, including two antigens that are differentially distributed on skeletal muscle fibers of the fast, fast/slow, and slow types. Monoclonal antibodies were prepared using partially purified membranes of adult chicken skeletal muscles as immunogens and were used to characterize three antigens associated with the sarcolemma of muscle fibers. Immunofluorescence staining of cryosections of adult and embryonic chicken muscles showed that two of the three antigens differed in expression by fibers depending on developmental age and whether the fibers were of the fast, fast/slow, or slow type. Fiber type was assigned by determining the content of fast and slow myosin heavy chain. MSA-55 was expressed equally by fibers of all types. In contrast, MSA-slow and MSA-140 differed in their expression by muscle fibers depending on fiber type. MSA-slow was detected exclusively at the periphery of fast/slow and slow fibers, but was not detected on fast fibers. MSA-140 was detected on all fibers but fast/slow and slow fibers stained more intensely suggesting that these fiber types contain more MSA-140 than fast fibers. These sarcolemma-associated antigens were developmentally regulated in ovo and in vitro. MSA-55 and MSA-140 were detected on all primary muscle fibers by day 8 in ovo of embryonic development, whereas MSA-slow was first detected on muscle fibers just before hatching. Those antigens expressed by fast fibers (MSA-55 and MSA-140) were expressed only after myoblasts differentiated into myotubes, but were not expressed by fibroblasts in cell culture. Each antigen was also detected in one or more nonskeletal muscle cell types: MSA-55 and MSA-slow in cardiac myocytes and smooth muscle of gizzard (but not vascular structures) and MSA-140 in cardiac myocytes and smooth muscle of vascular structures. MSA-55 was identified as an Mr 55,000, nonglycosylated, detergent-soluble protein, and MSA-140 was an Mr 140,000, cell surface protein. The Mr of MSA-slow could not be determined by immunoblotting or immunoprecipitation techniques. These findings indicate that muscle fibers of different physiological function differ in the components associated with the sarcolemma. While the function of these sarcolemma-associated antigens is unknown, their regulated appearance during development in ovo and as myoblasts differentiate in culture suggests that they may be important in the formation, maturation, and function of fast, fast/slow, and slow muscle fibers.

Abstract

There has been little or no integration of specific studies from the clinical literature with computer-based medical advice systems. This paper reports preliminary results of a research project designed to model reasoning from the clinical literature. The program, named "Roundsman", draws upon structured representations of the clinical literature in order to critique plans for medical management. This paper discusses the need for a clinical "distance" metric to use in mapping from studies to treatment choices. The design of one such metric is outlined, and the results of its incorporation in Roundsman are shown in a sample output from the program. The application domain for this program is the management of primary breast cancer, but the research goals are to identify general issues which arise in diverse medical management domains.

Abstract

This paper explores the premise that a formalized representation of empirical studies can play a central role in computer-based decision support. The specific motivations underlying this research include the following propositions: Reasoning from experimental evidence contained in the clinical literature is central to the decisions physicians make in patient care. A computational model, based upon a declarative representation for published reports of clinical studies, can drive a computer program that selectively tailors knowledge of the clinical literature as it is applied to a particular case. The development of such a computational model is an important first step toward filling a void in computer-based decision support systems. Furthermore, the model may help us better understand the general principles of reasoning from experimental evidence both in medicine and other domains. Roundsman is a developmental computer system which draws upon structured representations of the clinical literature in order to critique plans for the management of primary breast cancer. Roundsman is able to produce patient-specific analyses of breast cancer management options based on the 24 clinical studies currently encoded in its knowledge base. The Roundsman system is a first step in exploring how the computer can help to bring a critical analysis of the relevant literature to the physician, structured around a particular patient and treatment decision.

Abstract

Epithelial cell differentiation frequently occurs in situ in conjunction with supporting mesenchyme or connective tissue. In embryonic development the importance of the supporting mesenchyme for cytodifferentiation and morphogenesis has been demonstrated in several epithelial tissues, but the importance of epithelial-connective tissue interactions is less well studied in adult epithelial organs. We have investigated the interaction of adult mammary epithelial cells with adipocytes, which compose the normal supporting connective tissue in the mammary gland. Mammary epithelial cells from mice in various physiological states were cultured on cellular substrates of adipocytes formed from cells of the 3T3-L1 preadipocyte cell line. We found that there were two distinct phases to the interaction of epithelial cells with adipocytes. Cytodifferentiation of the epithelial cells and milk protein production were dependent on lactogenic hormones (insulin, hydrocortisone, and prolactin), whereas ductal morphogenesis was lactogenic hormone independent. When cultured on preadipocytes or adipocytes, mammary epithelial cells from never pregnant, pregnant, lactating, and involuting mice responded to lactogenic hormones rapidly by producing and secreting large amounts of alpha-, beta-, and gamma-casein and alpha-lactalbumin. This response was seen in individual as well as in clusters of epithelial cells, but was not seen if the same cells were cultured on tissue culture dishes without adipocytes, on fibroblasts (human newborn foreskin fibroblasts) or in the presence of adipocytes but in the absence of lactogenic hormones. Continued incubation of mammary epithelial cells on adipocytes in the presence or absence of lactogenic hormones resulted in the formation of a branching ductal system. Mammary epithelial cells in ducts that formed in the absence of lactogenic hormones produced no casein, but rapidly synthesized casein when subsequently exposed to these hormones. Ultrastructural studies revealed that the formation of a basement membrane occurs only in co-cultures of mammary epithelium with adipocytes or preadipocytes. Ultrastructural changes associated with secretion occurred only in the presence of lactogenic hormones. We propose that growth and formation of a ductal system in vitro can occur in the absence of lactogenic hormones, but that certain environment-associated events must occur if the epithelium is to become responsive to lactogenic hormones and undergo the cytodifferentiation associated with lactation.

Abstract

We show that a single myogenic progenitor cell in vitro generates two types of myoblasts committed to two distinct myogenic cell lineages. Using fast and slow myosin heavy chain isoform content to define myotube type, we found that myogenic cells from fetal quail (day 10 in ovo) formed two types of myotubes in vitro: fast and mixed fast/slow. Clonal analysis showed that these two types of myotubes were formed from two types of myoblasts committed to distinct fast and fast/slow lineages. Serial subcloning demonstrated that the initial myoblast progeny of an individual myogenic progenitor cell were in the fast lineage, whereas later progeny were in the fast/slow lineage. Fast and slow myosin expression within particular myotubes reflects the genetic processes underlying myoblast commitment to diverse myogenic lineages.

Abstract

The developmental regulation of myoblasts committed to fast, mixed fast/slow, and slow myogenic cell lineages was determined by analyzing myotube formation in high density and clonal cultures of myoblasts isolated from chicken and quail embryos of different ages. To identify cells of different myogenic lineages, myotubes were analyzed for content of fast and slow classes of myosin heavy chain (MHC) isoforms by immunocytochemistry and immunoblotting using specific monoclonal antibodies. Myoblasts from the hindlimb bud, forelimb bud, trunk, and pectoral regions of the early chicken embryo and hindlimb bud of the early quail embryo (days 3-6 in ovo) were committed to three distinct lineages with 60-90% of the myoblasts in the fast lineage, 10-40% in the mixed fast/slow lineage, and 0-3% in the slow lineage depending on the age and species of the myoblast donor. In contrast, 99-100% of the myoblasts in the later embryos (days 9-12 in ovo) were in the fast lineage. Serial subculturing from a single myoblast demonstrated that commitment to a particular lineage was stably inherited for over 30 cell doublings. When myoblasts from embryos of the same age were cultured, the percentage of muscle colonies of the fast, fast/slow, and slow types that formed in clonal cultures was the same as the percentage of myotubes of each of these types that formed in high density cultures, indicating that intercellular contact between myoblasts of different lineages did not affect the type of myotube formed. An analysis in vivo showed that three types of primary myotubes--fast, fast/slow, and slow--were also found in the chicken thigh at day 7 in ovo and that synthesis of both the fast and slow classes of MHC isoforms was concomitant with the formation of primary myotubes. On the basis of these results, we propose that in the avian embryo, there is an early phase of muscle fiber formation in which primary myotubes with differing MHC contents are formed from myoblasts committed to three intrinsically different primary myogenic lineages independent of innervation and a later phase in which secondary myotubes are formed from myoblasts in a single, secondary myogenic lineage with maturation and maintenance of fiber diversity dependent on innervation.

Abstract

We have examined the types of fast myosin heavy chains (MHCs) expressed in a number of different developing chicken skeletal muscles by combining peptide mapping and immunoblotting to identify fast MHC-specific peptides among the total mixture of MHC digestion products. Using this technique, we have identified three different fast MHC patterns among the different fast and mixed (i.e., fast and slow) fiber type muscles of the adult. While the different muscles all underwent sequential changes in fast MHC isoform expression during their development, the exact sequence of these changes and the isoform patterns expressed varied from muscle to muscle. During late embryonic or fetal development, all muscles expressed a similar fast MHC pattern (designated here as the fetal pattern) which was replaced shortly after hatching with a different fast MHC pattern (the neonatal pattern). During the transition from the neonatal to the adult state that occurred sometime in the first year after hatching, many of the muscles underwent additional changes in fast MHC isoform expression. In muscles such as the pectoralis major and pectoralis minor, a new fast MHC isoform pattern was seen in the adult so that the developmental program of isoform switching in these muscles involved the sequential appearance of distinct fetal, neonatal, and adult fast MHCs. Other muscles, such as the sartorius and posterior latissimus dorsi, underwent a qualitatively different program of isoform switching and expressed as an adult a fast MHC pattern that was indistinguishable from that expressed during fetal development. Finally, in some muscles, such as the superficial biceps, no change in isoform pattern was detected during the neonatal to adult transition--in these muscles, expression of the neonatal MHC isoform pattern apparently persisted into the adult state. These data indicate that no single scheme or program of fast MHC isoform switching can describe all the developmental changes that occur in fast MHC isoform expression in the chicken and that at least three different programs of isoform switching and expression can be identified.

DEVELOPMENTAL ORIGINS OF SKELETAL-MUSCLE FIBERS - CLONAL ANALYSIS OF MYOGENIC CELL LINEAGES BASED ON EXPRESSION OF FAST AND SLOW MYOSIN HEAVY-CHAINSPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMILLER, J. B., Stockdale, F. E.1986; 83 (11): 3860-3864

Abstract

A clonal analysis was used to show that skeletal muscle myoblasts are committed to distinct cell lineages during development. Myoblasts taken from embryonic chicken hindlimb muscles of different ages were cultured at clonal density. The content of fast and slow classes of the myosin heavy chain isoforms in the myotubes of the resulting muscle colonies was determined immunocytochemically with specific monoclonal antibodies that served as markers for the different fiber types. The muscle colonies formed by cloning myoblasts from early hindlimbs (days 4-6 in ovo) were of three types: the most numerous type, in which all myotubes in a colony contained only the fast class of myosin heavy chain; a less numerous type, in which all myotubes in a colony contained both the fast and slow classes of myosin heavy chain isoforms; and a rare type, in which all myotubes in a colony contained only the slow class of myosin heavy chain. The muscle colonies formed by cloning myoblasts from later hindlimbs (days 10-12 in ovo) were, however, all of one type, in which every myotube in a colony contained only fast myosin heavy chain. Thus, myoblasts in the early embryo (days 4-6 in ovo) were a heterogeneous population committed to three myogenic lineages: fast, mixed fast/slow, and slow, whereas myoblasts from the later embryo (days 10-12 in ovo) were only in the fast myogenic lineage. These results suggest that muscle fiber formation is rooted in two developmental phases--an early phase in which diverse fiber types are formed from intrinsically diverse populations of myoblasts and a later phase in which fibers are formed from a single population of myoblasts.

Abstract

Monoclonal antibodies specific to the light- and heavy-chain subunits of chicken skeletal muscle myosin have been used to identify fast and slow myosin-containing fibers in the thigh muscles of embryonic and adult chickens and to determine when in development diversification of muscle fiber types first occurs. Primary generation fibers which expressed different MLC and MHC types were evident within the dorsal and ventral premuscle masses and in the first muscles to form in the limb. These early embryonic muscle fiber types became distributed among and within the individual muscles of the thigh in a characteristic spatial pattern which served as a "blueprint" for guiding future muscle development and predicting the future fiber composition of the muscle. Despite the continuous addition of muscle fibers to the limb throughout development, the pattern remained unchanged. Neither the time of appearance, initial specialization, nor characteristic distribution of these primary fiber types within the limb was altered during the early embryonic period by chronic neuromuscular paralysis induced by D-tubocurarine. In contrast, muscles at later stages of embryonic development were markedly affected by such treatments and underwent atrophy and loss of differential staining characteristics. These results demonstrate that diversification of fibers in terms of myosin content is one of the earliest events in the formation of these muscles and suggest that the development of avian muscles be divided into two phases: an embryonic phase during which fibers of differing myosin content appear independently of innervation to become distributed in a specific topographic pattern within each muscle as it forms, followed by a fetal phase during which innervation becomes essential for maintaining this pattern and modulating the myosin content of its fibers.

Abstract

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.

Abstract

Mammary epithelium differentiates in a stromal milieu of adipocytes and fibroblasts. To investigate cell-cell interactions that may influence mammary epithelial cell differentiation, we developed a co-culture system of murine mammary epithelium and adipocytes and other fibroblasts. Insofar as caseins are specific molecular markers of mammary epithelial differentiation, rat anti-mouse casein monoclonal antibodies were raised against the three major mouse casein components to study this interaction. Mammary epithelium from mid-pregnant mice was plated on confluent irradiated monolayers of 3T3-L1 cells, a subclone of the Swiss 3T3 cell line that differentiates into adipocytes in monolayer culture and other cell monolayers (3T3-C2 cells, Swiss 3T3 cells, and human foreskin fibroblasts). Casein was synthesized by mammary epithelium only in the presence of co-cultured cells and the lactogenic hormone combination of insulin, hydrocortisone, and prolactin. Synthesis and accumulation of alpha-, beta-, and gamma-mouse casein within the epithelium was shown by immunohistochemical staining of cultured cells with anti-casein monoclonal antibodies, and the specificity of the immunohistochemical reaction was demonstrated using immunoblots. A competitive immunoassay was used to measure the amount of casein secreted into the culture medium. In a 24-h period, mammary epithelium co-cultured with 3T3-L1 cells secreted 12-20 micrograms beta-casein per culture dish. There was evidence of specificity in the cell-cell interaction that mediates hormone-dependent casein synthesis. Swiss 3T3 cells, newborn foreskin fibroblasts, substrate-attached material ("extracellular matrix"), and tissue culture plastic did not support casein synthesis, whereas monolayers of 3T3-L1 and 3T3-C2 cells, a subclone of Swiss 3T3 cells that does not undergo adipocyte differentiation, did. We conclude that interaction between mammary epithelium and other specific nonepithelial cells markedly influences the acquisition of hormone sensitivity of the epithelium and hormone-dependent differentiation.

Abstract

Murine mammary epithelium grows in association with predominantly adipocyte stroma in vivo. To investigate potential growth-promoting effects of adipocytes on mammary epithelium, we developed a co-culture system of mammary epithelium and adipocytes by taking advantage of the 3T3-L1 cell line. These cells undergo adipocyte differentiation when the culture reaches confluence and growth ceases. Mid-pregnant murine mammary epithelium was plated on lethally irradiated feeder layers of 3T3-L1 adipocytes, undifferentiated 3T3-L1 cells, 3T3-C2 fibroblasts (a subclone of 3T3 cells that does not undergo adipocyte differentiation), or tissue culture plastic. Mammary epithelial colony size on adipocyte feeder layers was 2-fold larger than colonies on 3T3-C2 cells and 4-fold larger than colonies on tissue culture plastic. Measurement of tritiated thymidine [3H]TdR incorporation and labelling index in mammary cells was significantly higher on adipocytes than on other feeder layers or plastic. There was a 6-fold increase in mammary cell number after 5 days in culture when mammary epithelium was plated on substrate-attached material ('extracellular matrix') derived from 3T3-L1 cells and a 4-fold increase in cell number when plated on plastic in conditioned medium derived from 3T3-L1 adipocytes compared with growth on plastic in unconditioned medium. We conclude that interaction of mammary epithelium with adipocytes results in a marked increase in proliferation of mammary epithelium and that extracellular components may mediate this effect.

Abstract

The dose of adjuvant chemotherapy for breast cancer may be an important factor in the success of the treatment program. In a retrospective analysis, the authors determined whether patients who were irradiated either postoperatively (N = 29) or as part of primary treatment (N = 13) received a lower dose of adjuvant cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) chemotherapy than patients who were not irradiated (N = 42). The 84 evaluable patients received either cyclical or weekly CMF. Radiation therapy included the chest wall or breast and regional lymph nodes. The mean percentage of maximum chemotherapy dose delivered (59.9% versus 73.5%; P less than 0.001), mean percent prescribable or theoretical maximum dose (83.1% versus 91.3%; P less than 0.001), and mean leukocyte count (3.9 versus 4.5; P less than 0.01) during therapy were statistically significantly lower in irradiated patients. The lower delivered chemotherapy dose in irradiated patients was not related to the radiation dose to the thoracic spine. The authors conclude that radiation therapy to the chest wall or breast and regional lymph nodes reduces the dose of adjuvant CMF that can be delivered.

Abstract

Endomyocardial biopsy was done 119 times in 98 patients receiving doxorubicin therapy once every 3 weeks and 41 times in 27 patients receiving doxorubicin therapy weekly. Factors contributing to the degree of anthracycline-induced endomyocardial injury were evaluated. Neither age, sex, type of malignancy, concomitant use of other chemotherapeutic agents including cyclophosphamide, nor history of cardiac disease or hypertension influenced the extent of the endomyocardial injury. The dose of doxorubicin (p = 0.0001) and the schedule (weekly versus 3 weekly) (p = 0.0020) independently predicted the degree of endomyocardial damage in multivariate analyses. Previous cardiac irradiation had borderline significance (p = 0.074) in predicting endomyocardial damage in this analysis. Doxorubicin therapy administered on a weekly schedule is associated with less anthracycline-induced cardiac damage than is doxorubicin therapy delivered in the conventional, 3-weekly schedule.

Abstract

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.

Abstract

Two prospective, controlled studies were conducted to determine if psychological and social functioning could be enhanced in patients with Hodgkin's disease by either education or participation in a peer support therapy group. Eighty-one patients were evaluated with the Cancer Patient Behavior Scale prior to and following intervention. Following education, patients experienced significant improvement in the frequency of Anxiety, Treatment Problems, Depression, and Life Disruption (p less than or equal to 0.1) compared to a control group. Following participation in peer support groups, patients showed no improvement in any of 10 areas of life functioning. Thus education represents an effective, efficient, and inexpensive means of improving psychological and social behavior in patients with Hodgkin's disease. Participation in this peer support therapy group did not result in significant behavior change.

Abstract

Myoblasts from 12-day chick embryos in cell culture transport the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB) two to three-fold more rapidly than multinucleated myotubes which form from them. This decrease in transport is due to a relative decrease in the number of transport sites per unit area of cell surface suggesting a compositional change in the plasma membrane during myogenesis. In studies reported here, AIB transport was monitored throughout myogenesis and correlated with other aspects of differentiation. During myogenesis the number of amino acid transport sites remains constant per myotube nucleus. As myogenesis proceeds, there is a marked increase in cellular protein and cell surface without a commensurate increase in amino acid transport sites. The net consequence of the surface area change is fewer amino acid transport sites per unit area of myotube membrane surface. The decrease in membrane transport sites for AIB per unit area of membrane is not a result of length of time in culture per se, medium depletion, or cell density, but is a result of differentiation, since blocking myoblast fusion by deprivation of calcium delays the decrease in AIB transport sites per unit cell surface area while reversal of the calcium deprivation block is accompanied by a rapid decrease in the number of AIB transport sites per unit cell surface area. Thus, the decrease in AIB transport sites is an aspect of differentiation which accompanies the marked elaboration of surface membrane during myogenesis.

Abstract

The differentiation of skeletal muscle is characterized by recognition, alignment, and subsequent fusion of myoblast cells at their surfaces to form large, multinucleated myotubes. Monoclonal antibodies were used to investigate antigenic changes in the cell surface membrane specific for various stages of myogenesis. Chick embryonic skeletal muscle cells were cultured in vitro to the desired stage of differentiation and then injected into BALB/c mice. Spleen cells from the immunized mice were hybridized with NS-1 or P3 8653 mouse myeloma cells. Hybrid cell clones were selected in HAT medium and screened using an indirect radioimmunoassay for the production of monoclonal antibodies specific to myogenic cell surfaces. Target cells for the radioimmunoassay included three stages of myogenesis (myoblasts, midfusion, myoblasts, and myotubes) and chick lung cells as a control for polymorphic antigens. Sixty-one clones were obtained which produced antibodies specific for myogenic cells. Thirty-five of these clones were generated from mice immunized with midfusion myoblast stages of myogenesis and 26 were obtained from mice immunized with the later myotube stage of myogenesis. Quantitative measurements by RIA of myogenic determinants per cell surface area on each target cell type revealed that most of the determinants decrease during myogenesis when midfusion myoblasts are used as the immunogen. When myotube stages are used as the immunogen, more determinants increase with cell differentiation. Therefore, the most common pattern of determinant change is for them to be present at all stages of myogenesis but to vary quantitatively through development. There are determinants unique to each stage of myogenesis and marked quantitative differences within a cell stage for each determinant.

MYOSIN LIGHT-CHAINS AND THE DEVELOPMENTAL ORIGIN OF FAST MUSCLEPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCESStockdale, F. E., Raman, N., Baden, H.1981; 78 (2): 931-935

Abstract

Physiological characteristics of embryonic and fetal fast muscle function are similar to those of adult slow muscles, whereas most biochemical data suggest that embryonic and fetal fast muscles contain only fast muscle myosin. In the studies reported here, myofibrillar preparations from developing avian pectoral muscle (fast muscle) were isolated and analyzed for myosin light-chain type and synthesis. These analyses show that early in development avian fast muscle synthesizes and assembles myofibrils with light chains of both slow and fast myosins. Later in development, fast muscle no longer assembles myofibrils containing slow myosin light chains due to the cessation of synthesis of slow myosin light chains in mid-development. These in vivo studies indicate that the more developmentally primitive type of skeletal muscle is one that synthesizes both slow and fast myosin light chains independent of its anatomic location, and an event(s) late in fast muscle development results in the repression of synthesis of slow myosin light chains.

Abstract

Serum and tumor tissue from breast cancer patients were evaluated for the presence of alpha subunit of hCG, by means of the specific radioimmunoassay. Elevated serum levels occurred in 0/50 (0%) of normal female controls, 1/17 (6%) of patients with benign breast disease, 3/53 (6%) of patients with primary breast cancer, and 12/84 (14%) of patients with metastatic breast cancer. Cytosols from 10/56 (18%) primary tumors and 10/37 (28%) metastatic tumors were found to contain the alpha subunit. Comparison of serum and tumor cytosol levels in nine patients with metastatic disease showed a correspondence between elevated cytosol levels and elevated serum levels. No similar correspondence in 25 patients with primary disease was observed. Alpha subunit was isolated from one tumor cytosol and was characterized with respect to its immunochemical cross-reactivity and molecular weight as compared with native alpha subunit from hCG. For both parameters, the native and ectopic alpha subunit were found to be identical.

Abstract

The growth promoting effects of lithium and insulin on cultures of mammary gland epithelium and CZF mouse mammary tumor cells were investigated. Lithium chloride exerts a 450-fold increase in the rate of DNA synthesis in mammary epithelium from mid-pregnant mice in organ culture or monolayer culture. There is an increase in both the percentage of cells initiating DNA synthesis and the net accumulation of DNA. The most effective lithium concentration is 10 mM, and the maximally effective rate of stimulation is reached 48 hours after addition. The magnitude of response to lithium varies with the physiological state of the mammary epithelial cell donor: epithelium from non-pregnant or lactating mice is less responsive than that from mid-pregnant mice. In combination, insulin and lithium produce either a synergistic or an additive effect on the growth of epithelium dependent upon the physiological state of the donor animal. Lithium also promotes the growth of mammary tumor cells in the absence or serum or other mitogens. The action of lithium on DNA synthesis appears to be a direct effect on the epithelial cells.

Abstract

This report describes the partial purification and characterization of mammary stimulating factor (MSF), a mitogenic peptide isolated from serum which initiates growth in mouse mammary epithelium. By using ion-exchange chromatography, gel filtration, and isoelectric focusing, MSF was purified 250-fold from porcine serum. It is a heat-stable protein of molecular weight 10,100--10,400 with an isoelectric point of 5.5--6.0. MSF initiates DNA synthesis in vitro in mammary epithelium to a greater extent than in mouse mammary tumor cells (CZF), 3T3 cells, or chick embryo cells. Comparison of the biological, physical, and immunological properties of MSF with other established growth-promoting peptides suggests that MSF is a unique serum factor.

Abstract

When cultures of skeletal muscle cells of the chick embryo are subjected to repetitive, electrical stimulation, the contractions increase the amount of protein produced by these cells. The increase is greater for contractile proteins such as myosin heavy chain than for total cellular protein. This demonstrates that in a culture system of skeletal muscle cells that have differentiated in the absence of innervation, one can elicit the protein synthetic response associated with skeletal muscle hypertrophy in vivo.

Abstract

The proliferative response of mammary gland epithelium from nonpregnant, pregnant, and lactating mice to mammary serum factor and insulin was studied in vitro. Mammary gland epiithelium from nonpregnant and lactating animals has a delayed proliferative response to mammary serum factor and insulin when compared to the response of epithelium from pregnant animals. The results show that as the animals go through pregnancy into lactation the mammary gland epithelium becomes less responsive to mammary serum factor while it retains its responsiveness to insulin. The concentration of mammary serum factor in sera from animals at various physiological stages is constant. Sera from hypophysectomized rats, on the other hand, show a 50% drop in mammary serum factor activity. This loss of activity cannot be reversed by injecting prolactin, 17-beta-estradiol, or growth hormone into the hypophysectomized animals. A hypothesis that the mammary gland is composed of two proliferative epithelial populations is developed, and the possible role of prolactin in stimulating DNA synthesis is discussed.

Abstract

During development of the limb musculature there is an eclipse of the myoblasts from the time first formed until the limb musculature begins to develop. The formation of the muscles of the limb is from cells of somite origin that migrate into the forming limb bud. While it is possible to identify myogenic cells in the somite prior to when migration occurs, and to determine the time frame during which migration occurs, there is a hiatus of about 34 hours during which it is difficult to identify or culture myogenic cells from the limb. In part this is because it is difficult to distinguish myogenic cells that migrate from those that do not. There are no markers that distinguish the myogenic cells of the somite that form the axial muscles and therefore stay in place, and from the myogenic cells that migrate to form the peripheral muscles of the body. Thus it unclear if the cell commitment to different myogenic fates occurs during somite formation, or during their sojourn to the limb bud.